Antitumor T-cell Research Articles

Benzosceptrin C induces lysosomal degradation of PD-L1 and promotes antitumor immunity by targeting DHHC3

Programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) blockade has become a mainstay of cancer immunotherapy. Targeting the PD-1/PD-L1 axis with small molecules is an attractive approach to enhance antitumor immunity. Here, we identified a natural marine product, benzosceptrin C (BC), that enhances the cytotoxicity of Tcells to cancer cells by reducing the abundance of PD-L1. Furthermore, BC exerts its antitumor effect in mice bearing MC38 tumors by activating tumor-infiltrating Tcell immunity. Mechanistic studies suggest that BC can prevent palmitoylation of PD-L1 by inhibiting DHHC3 enzymatic activity. Subsequently, PD-L1 is transferred from the membrane to the cytoplasm and cannot return to the membrane via recycling endosomes, triggering lysosome-mediated degradation of PD-L1. Moreover, the combination of BC and anti-CTLA4 effectively enhances antitumor Tcell immunity. Our findings reveal a previously unrecognized antitumor mechanism of BC and represent an alternative immune checkpoint blockade (ICB) therapeutic strategy to enhance the efficacy of cancer immunotherapy.

Abstract B017: NINJA PDAC: A robust murine pancreatic cancer organoid transplant model with inducible neoantigens for studying tumor microenvironment interactions with anti-tumor T-cell immunity

Abstract Pancreatic ductal adenocarcinoma (PDAC) is highly lethal due to early metastasis and poor therapeutic response including to anti-PD1/CTLA4-based immunotherapy, characteristics attributed to a desmoplastic tumor microenvironment (TME) and low immunogenicity. However, long-term human PDAC survivors exhibit higher neoantigen expression with active T-cell immunogenicity, and most human PDAC express predicted neoepitopes, arguing that T-cell mediated anti-tumor immunity shapes PDAC progression. By contrast, tumors in classical pre-clinical (KRASLSL-G12D; TRP53F/F; pancreas-specific Cre, or KPC) murine models exhibit low neoantigen burden, are non-immunogenic, with growth kinetics independent of T cells, making them poorly suited to study T-cell immunity. Combined chemoimmunotherapy regimens for human PDAC might be developed more efficiently via preclinical studies in murine models with physiologic stroma, defined neoantigens that can be induced in established tumors, and measurable neoantigen-specific T-cell responses. We developed NINJA PDAC, a novel, robust transplantable murine pancreatic cancer organoid model, to study neoantigen-specific anti-tumor T-cell responses and how they are influenced by the TME. The NINJA (Damo et al., 2021) system allows for spatially (Cre) and temporally (doxycycline+tamoxifen) controlled inducible expression of LCMV-derived neoantigens with low-to-moderate MHCI/II-binding affinity (GP33 and GP66, respectively) comparable to human PDAC, in Kras-mutated, p53-deficient PDAC organoids. Transplanted NINJA PDAC organoids engraft robustly both subcutaneously and orthotopically in syngeneic mice, with reproducible kinetics. Unlike 2D cell line transplants, these organoid-derived tumors have a complex ductal architecture, immune islands, and fibrotic stroma more representative of human PDAC. T-cell depletion studies demonstrated that full rejection of tumors with constitutive neoantigen expression (NINJA ‘ON’) requires both CD4+ and CD8+ neoantigen-specific T-cells, allowing the study of the roles of both helper and cytotoxic T-cells in anti-PDAC immunity. ‘Poised’ NINJA PDAC organoids do not express neoantigen until induction, and we confirmed high tumor neoantigen expression can be induced with doxycycline/tamoxifen in vivo (38%±18% neoantigen-expressing tumor cells). Early induction in vivo leads to intratumoral CD3+ T-cell infiltration, with increased neoantigen-specific (H2Db/gp33-41-tetramer positive) T-cells in tumors and tumor-draining lymph nodes. Late induction in vivo leads to neoantigen induction, but more muted T-cell infiltration and minimally attenuated tumor growth, suggesting a role of an established TME in suppressing T-cell responses. NINJA PDAC is a promising model that affords a high-fidelity toolset to study how murine neoantigen-specific anti-tumor T-cell responses are perturbed by the TME and further altered by host variables (such as obesity) that modulate the TME with the potential to expedite the development of new combination immunotherapies. Citation Format: Jeremy B. Jacox, Gena G. Foster, Dhruvi Shah, Nikhil S. Joshi, Mandar D. Muzumdar. NINJA PDAC: A robust murine pancreatic cancer organoid transplant model with inducible neoantigens for studying tumor microenvironment interactions with anti-tumor T-cell immunity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B017.

Abstract B079: Identification of synergistic drug combinations including MEK inhibitor and agents modulating the tumor microenvironment in an electroporation-induced pancreatic cancer model

Abstract Pancreatic ductal adenocarcinoma (PDAC) is refractory to cytostatic and immuno-oncology therapeutic strategies, leaving surgical resection of the tumor as the only treatment that can significantly prolong survival. We are therefore exploring optimized treatment strategies targeting cancer cells as well as infiltrating immune cells and the tumor microenvironment (TME) in an autochthonous mouse model for PDAC. Single focal tumors are induced through in vivo electroporation of gene constructs into the pancreas which encode mutated Kras and facilitate knock out the tumor suppressor genes P53, Cdkn2a and/or Smad4 via CRISPR/Cas9. The resulting tumors recapitulate the human disease with respect to genetics, histology and TME composition. Furthermore, this model allows the generation of tumors reflecting different PDAC subtypes, as well as the resection of primary tumors in combination with the (neo-)adjuvant regimens. Like the human disease, these tumors are refractory to chemotherapy and immune checkpoint blockade, whereas targeted therapy by means of MEK inhibitors merely leads to temporary delay of tumor outgrowth. We therefore tested the combination of MEK inhibitor treatment with different, potentially complementary modalities. We found the combination with agonist anti-CD40 antibodies to be highly effective. Prominent regressions are observed, and in combination with primary tumor resection long-term complete responses can be achieved even in a metastatic variant of the model. Apart from the direct cytostatic impact of MEKi on the Kras-transformed tumor cells, the mechanism of action of this synergistic drug combination involves enhancement of the anti-tumor T-cell response, suppression of CD4+ Tregs, and reduction of MDSCs and M2-type macrophages in favor of M1-type pro-inflammatory macrophages. A second highly effective regimen as identified in our experiments involves the combination of MEK inhibitors with the multi-kinase inhibitor regorafenib. In contrast to the aforementioned regimen, the complementary action of these drugs involves modulation of the TME by regorafenib. While the clinical translation of the MEKi/CD40 Ab regimen is hampered by toxicity issues encountered with agonist anti-CD40 Ab, the synergy as observed in the MEKi/regorafenib regiment provides a tangible path towards clinical testing. Citation Format: Aline S. Konrad, Tanja Hägele, Stefan Zens, Caroline Vent, Moritz Hamberger, Sarah Baiker, Aron Maes, Carl-Stephan Leonhardt, Oliver Strobel, Oliver Politz, Dieter Zopf, Daniel Baumann, Julian Mochayedi, Rienk Offringa. Identification of synergistic drug combinations including MEK inhibitor and agents modulating the tumor microenvironment in an electroporation-induced pancreatic cancer model [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B079.

Modified Dendritic cell-based T-cell expansion protocol and single-cell multi-omics allow for the selection of the most expanded and in vitro-effective clonotype via profiling of thousands of MAGE-A3-specific T-cells.

Adoptive cell therapy using TCR-engineered T-cells is one of the most effective strategies against tumor cells. The TCR T-cell approach has been well tested against a variety of blood neoplasms but is yet to be deeply tested against solid tumors. Among solid tumors, cancer-testis antigens are the most prominent targets for tumor-specific therapy, as they are usually found on cells that lie behind blood-tissue barriers. We have employed a novel efficient protocol for MAGE-A3-specific T-cell clonal expansion, performed single-cell multi-omic analysis of the expanded T-cells via BD Rhapsody, engineered a selected T-cell receptor into a lentiviral construct, and tested it in an in vitro LDH-cytotoxicity test. We have observed a 191-fold increase in the MAGE-A3-specific T-cell abundance, obtained a dominant T-cell receptor via single-cell multi-omic BD Rhapsody data analysis in the TCRscape bioinformatics tool, and observed potent cytotoxicity of the dominant-clonotype transduced TCR T-cells against a MAGE-A3-positive tumor. We have demonstrated the efficiency of our T-cell enrichment protocol in obtaining potent anti-tumor T-cells and their T-cell receptors, especially when paired with the modern single-cell analysis methods.

Mycobacterium smegmatis enhances shikonin-induced immunogenic cell death-an efficient in situ tumor vaccine strategy.

Tumor vaccines are a promising avenue in cancer immunotherapy. Despite the progress in targeting specific immune epitopes, tumor cells lacking these epitopes can evade the treatment. Here, we aimed to construct an efficient in situ tumor vaccine called Vac-SM, utilizing shikonin (SKN) to induce immunogenic cell death (ICD) and Mycobacterium smegmatis as an immune adjuvant to enhance in situ tumor vaccine efficacy. SKN showed a dose-dependent and time-dependent cytotoxic effect on the tumor cell line and induced ICD in tumor cells as evidenced by the CCK-8 assay and the detection of the expression of relevant indicators, respectively. Compared with the control group, the in situ Vac-SM injection in mouse subcutaneous metastatic tumors significantly inhibited tumor growth and distant tumor metastasis, while also improving survival rates. Mycobacterium smegmatis effectively induced maturation and activation of bone marrow-derived dendritic cells (DCs), and in vivo tumor-draining lymph nodes showed an increased maturation of DCs and a higher proportion of effector memory T-cell subsets with the Vac-SM treatment, based on flow cytometry analysis results. Collectively, the Vac-SM vaccine effectively induces ICD, improves antigen presentation by DCs, activates a specific systemic antitumor T-cell immune response, exhibits a favorable safety profile, and holds the promise for clinical translation for local tumor immunotherapy.

Programming peptide-oligonucleotide nano-assembly for engineering of neoantigen vaccine with potent immunogenicity.

Background: Neoantigen nanovaccine has been recognized as a promising treatment modality for personalized cancer immunotherapy. However, most current nanovaccines are carrier-dependent and the manufacturing process is complicated, resulting in potential safety concerns and suboptimal codelivery of neoantigens and adjuvants to antigen-presenting cells (APCs). Methods: Here we report a facile and general methodology for nanoassembly of peptide and oligonucleotide by programming neoantigen peptide with a short cationic module at N-terminus to prepare nanovaccine. The programmed peptide can co-assemble with CpG oligonucleotide (TLR9 agonist) into monodispersed nanostructures without the introduction of artificial carrier. Results: We demonstrate that the engineered nanovaccine promoted the codelivery of neoantigen peptides and adjuvants to lymph node-residing APCs and instigated potent neoantigen-specific T-cell responses, eliciting neoantigen-specific antitumor immune responses with negligible systemic toxicity. Furthermore, the antitumor T-cell immunity is profoundly potentiated when combined with anti-PD-1 therapy, leading to significant inhibition or even complete regression of established melanoma and MC-38 colon tumors. Conclusions: Collectively, this work demonstrates the feasibility and effectiveness of personalized cancer nanovaccine preparation with high immunogenicity and good biosafety by programming neoantigen peptide for nanoassembly with oligonucleotides without the aid of artificial carrier.

A trispecific antibody induces potent tumor-directed T-cell activation and antitumor activity by CD3/CD28 co-engagement.

Aim: A novel CD19xCD3xCD28 trispecific antibody with a tandem single-chain variable fragments (scFv) structure was developed for the treatment of B-cell malignancies. Methods: The trispecific antibody in inducing tumor-directed T-cell activation and cytotoxicity was evaluated in vitro and in vivo and compared with its bispecific counterpartBiTE-CD19xCD3lacking aCD28-targeting domain. Results: The trispecific antibody with aco-stimulatory domain exhibited augmented T-cell activation and memory T-cell differentiation capability and itinduced faster tumor cell lysis than the bispecific antibody. RNAseq analysis revealed that the trispecific antibody modulates CD3/TCR complex-derived signal and upregulates antiapoptotic factors to influence thesurvival of Tcells. Conclusion: By CD3/CD28 co-engagement, the trispecific antibody demonstrated its advantages in T-cell immunity and potential use as a more powerful and long-lasting T-cell engager.

Nanocodelivery of an NIR photothermal agent and an acid-responsive TLR7 agonist prodrug to enhance cancer photothermal immunotherapy and the abscopal effect

The immunosuppressive tumor microenvironment (TME) greatly limits the actual outcome of immunotherapy. Therefore, it is urgent to develop appropriate strategies to reshape the TME and ultimately induce a strong immune response. Here, we developed a dual-functional liposome loaded with the photothermal agent IR808 near the infrared region (NIR) and Toll-like-receptor-7 (TLR7) agonist loxoribine prodrug (Lipo@IR808@Loxo) to achieve NIR light-triggered photothermal therapy (PTT) and the targeted delivery of immune adjuvants. Under NIR irradiation, Lipo@IR808@Loxo could greatly improve the efficiency of PTT to directly kill tumor cells and release tumor-associated antigens, which could work together with loaded loxoribine to relieve the immunosuppressive TME, effectively promoting the activation of antigen-presenting cells and subsequent antigen presentation. In this way, Lipo@IR808@Loxo could act as an in situ therapeutic cancer vaccine, eventually inducing a potent antitumor T-cell response. When further combined with immune checkpoint blockade, Lipo@IR808@Loxo-mediated photothermal immunotherapy could not only eliminate the primary tumors but also inhibit the growth of distant tumors, thus enhancing the abscopal effect.

Single-Cell Transcriptomics Reveals the Heterogeneity of the Immune Landscape of IDH-Wild-Type High-Grade Gliomas.

Isocitrate dehydrogenase (IDH)-wild-type (WT) high-grade gliomas, especially glioblastomas, are highly aggressive and have an immunosuppressive tumor microenvironment. Although tumor-infiltrating immune cells are known to play a critical role in glioma genesis, their heterogeneity and intercellular interactions remain poorly understood. In this study, we constructed a single-cell transcriptome landscape of immune cells from tumor tissue and matching peripheral blood mononuclear cells (PBMC) from IDH-WT high-grade glioma patients. Our analysis identified two subsets of tumor-associated macrophages (TAM) in tumors with the highest protumorigenesis signatures, highlighting their potential role in glioma progression. We also investigated the T-cell trajectory and identified the aryl hydrocarbon receptor (AHR) as a regulator of T-cell dysfunction, providing a potential target for glioma immunotherapy. We further demonstrated that knockout of AHR decreased chimeric antigen receptor (CAR) T-cell exhaustion and improved CAR T-cell antitumor efficacy both in vitro and in vivo. Finally, we explored intercellular communication mediated by ligand-receptor interactions within the tumor microenvironment and PBMCs and revealed the unique cellular interactions present in the tumor microenvironment. Taken together, our study provides a comprehensive immune landscape of IDH-WT high-grade gliomas and offers potential drug targets for glioma immunotherapy.

The BCL-2 inhibitor APG-2575 resets tumor-associated macrophages toward the M1 phenotype, promoting a favorable response to anti-PD-1 therapy via NLRP3 activation

The main challenges in the use of immune checkpoint inhibitors (ICIs) are ascribed to the immunosuppressive tumor microenvironment and the lack of sufficient infiltration of activated CD8+ T cells. Transforming the tumor microenvironment (TME) from “cold” to “hot” and thus more likely to potentiate the effects of ICIs is a promising strategy for cancer treatment. We found that the selective BCL-2 inhibitor APG-2575 can enhance the antitumor efficacy of anti-PD-1 therapy in syngeneic and humanized CD34+ mouse models. Using single-cell RNA sequencing, we found that APG-2575 polarized M2-like immunosuppressive macrophages toward the M1-like immunostimulatory phenotype with increased CCL5 and CXCL10 secretion, restoring T-cell function and promoting a favorable immunotherapy response. Mechanistically, we demonstrated that APG-2575 directly binds to NF-κB p65 to activate NLRP3 signaling, thereby mediating macrophage repolarization and the activation of proinflammatory caspases and subsequently increasing CCL5 and CXCL10 chemokine production. As a result, APG-2575-induced macrophage repolarization could remodel the tumor immune microenvironment, thus improving tumor immunosuppression and further enhancing antitumor T-cell immunity. Multiplex immunohistochemistry confirmed that patients with better immunotherapeutic efficacy had higher CD86, p-NF-κB p65 and NLRP3 levels, accompanied by lower CD206 expression on macrophages. Collectively, these data provide evidence that further study on APG-2575 in combination with immunotherapy for tumor treatment is required.

Genetically engineered eukaryocyte-bacteria hybrid membrane-camouflaged nanoemulsion for three-pronged synergistic cancer therapy

Despite the tremendous clinical benefits of immune checkpoint blockade (ICB), it still suffers from several drawbacks such as low response rate, off-target toxicity, and high costs. To address these issues, herein, a hybrid membrane (PSHM) consisting of outer membrane vesicles of attenuated Salmonella typhimurium (SM) and the membrane of PD-1-expressing HEK293T cells (PM) is fabricated to coat nanoemulsion for three-pronged synergistic photothermal-immunotherapy. First, SM upregulates the PD-L1 expression of tumor cells to enhance the response rate of PD-L1 blockade. Second, SM targets the nanoemulsion to the hypoxic tumor to realize targeted PD-L1 blockade mediated by PD-1 proteins on the PM. Third, SM induces erythrocyte extravasation into tumors for photothermal ablation of tumors without damaging surrounding tissues and thereby enhancing anticancer immunity. The hybrid membrane-coated nanoemulsion arouses a potent antitumor T-cell immunity and inhibits tumor growth and metastasis. Therefore, this study presents a promising hybrid membrane coating strategy to potentiate ICB.

De novo NAD+ synthesis contributes to CD8+ T cell metabolic fitness and antitumor function

The dysfunction and clonal constriction of tumor-infiltrating CD8+ Tcells are accompanied by alterations in cellular metabolism; however, how the cell-intrinsic metabolic pathway specifies intratumoral CD8+ Tcell features remains elusive. Here, we show that cell-autonomous generation of nicotinamide adenine dinucleotide (NAD+) via the kynurenine pathway (KP) contributes to the maintenance of intratumoral CD8+ Tcell metabolic and functional fitness. De novo NAD+ synthesis is involved in CD8+ Tcell metabolism and antitumor function. KP-derived NAD+ promotes PTEN deacetylation, thereby facilitating PTEN degradation and preventing PTEN-dependent metabolic defects. Importantly, impaired cell-autonomous NAD+ synthesis limits CD8+ Tcell responses in human colorectal cancer samples. Our results reveal that KP-derived NAD+ regulates the CD8+ Tcell metabolic and functional state by restricting PTEN activity and suggest that modulation of de novo NAD+ synthesis could restore CD8+ Tcell metabolic fitness and antitumor function.

Abstract A013: Targeting perivascular macrophages with an orally bioavailable heme oxygenase-1 inhibitor improves responses to chemotherapeutic drugs in cancer

Abstract The tumor microenvironment (TME) plays a crucial role in cancer progression. It consists of various elements including stromal cells, proteins, and physical factors that support the growth and spread of cancer. Targeting the TME has become an appealing strategy for cancer treatment. Tumor-associated macrophages (TAMs) are an abundant stromal cell type in the TME and, due to their plasticity, TAMs can be exploited by cancer cells and the broader stromal reaction to promote a pro-tumoral response. TAMs have been implicated in facilitating a variety of pro-tumoral pathways including resistance to anti-cancer therapeutics, immune suppression, angiogenesis, and metastasis. Our group has recently characterized a subpopulation of pro-angiogenic and immune-suppressive TAMs which can be identified by their expression of the lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1). LYVE-1+ TAMs reside spatially proximal to blood vasculature and adopt a collaborative multi-cellular ‘nest’ arrangement which may support their biological activity. LYVE-1+ TAMs selectively express high levels of the immunomodulatory enzyme heme oxygenase-1 (HO-1), an enzyme which catabolizes heme to generate the biologically active catabolites carbon monoxide (CO), biliverdin and ferrous iron. Genetic inactivation of HO-1 in LYVE-1+ TAMs results in improved anti-tumoral CD8 T-cell responses and enhanced response to chemotherapy, resulting in prolonged tumor control in a spontaneous MMTV-PyMT murine model of breast cancer. Unfortunately, current HO-1 inhibitors are not orally bioavailable, which limits their utility as immunotherapeutics for the treatment of cancer. In response to this hurdle, we have developed a next generation HO-1 inhibitor called KCL-HO-1i. We demonstrate in pharmacokinetic studies that KCL-HO-1i is orally bioavailable in murine models with a serum half-life of around 3hours. Furthermore, orally delivering KCL-HO-1i alongside standard of care chemotherapy is able to deliver durable tumor control in MMTV-PyMT mice. We have characterized the response of the TME to KCL-HO-1i using flow cytometry, immunofluorescence and RNA sequencing approaches and demonstrate that KCL-HO-1i supports a broader switch from an immunological ‘cold’ to ‘hot’ TME which provides a more favourable immune landscape for improving the response to chemotherapeutic drugs. Taken together, our data support the use of KCL-HO-1i as novel immunotherapeutic for the treatment of cancer. Citation Format: Meriem Bahri, Taha Al-Adhami, Emre Demirel, Joanne E. Anstee, Karen T. Feehan, James Rosekilly, Cheryl E. Gillett, Dominika Sosnowska, Renee Gitsaki-Taylor, Tik Shing Cheung, James F. Spicer, Khondaker Miraz Rahman, James N. Arnold. Targeting perivascular macrophages with an orally bioavailable heme oxygenase-1 inhibitor improves responses to chemotherapeutic drugs in cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A013.

Abstract C083: HDAC6 inhibition increases proteasome activity and modulates the myeloma immunopeptidome to promote cytotoxic T-cell activity

Abstract The proteasome functions as an essential component of immune surveillance by generating peptides from intracellular antigens that are then presented to cytotoxic T-cells. The vast majority of defined antitumor T-cell responses involves the proteasomal processing of intracellular proteins and their presentation in the context of major histocompatibility complex (MHC) class I molecules to antigenic peptide-specific CD8+ T-cells. Such antigenic peptides generated by the proteasome are generally 8-10 amino acids in length and mirror the linear sequence of the parental protein. However, immune evasion is a hallmark of cancer cells that disrupt the antigen processing and presentation machinery. Tumors develop strategies to evade host immunity and resist current immunotherapies, while others display a low tumor mutation burden limiting the presence of tumor antigenicity. Here, we postulated that pharmacologic enhancement of proteasome catalytic activity would increase MHC class I antigen presentation, modulate the myeloma immunopeptidome, and promote CD8+ T-cells anti-myeloma activity. A cell-based, high-throughput screen revealed that the histone deacetylase 6 (HDAC6) inhibitors tubastatin-A, ACY-738 and ACY-1215 increased proteasome activity in multiple myeloma (MM) cells. HDAC6 inhibitors increased pan-MHC class-I expression in MM cells as well as a panel of cancer cell lines. Treatment of MM patient tumor cells with HDAC6 inhibitors increased the cytotoxic effect of autologous patient-derived T-cells. Mechanism-based studies demonstrated that pharmacologic blockade or genetic ablation of the HDAC6 ubiquitin-binding (BUZ) domain disrupted HDAC6 association with HR23B, a substrate-shuttle factor that delivers ubiquitinylated cargo to proteasomes. HDAC6 inhibitors or HDAC6-BUZ domain deletion reduced the association of HR23B with HDAC6 and promoted binding of HR23B to proteasomes and a consequent increase in proteasome activity. The results validate the HDAC6-BUZ domain as an attractive drug discovery target and demonstrate that proteasome activators enhance antitumor immunity. Proteasome activators represent a paradigm-shifting approach to overcome mechanisms of immune escape in cancer cells. FDA-approved drugs that increase proteasome activity and boost antigen presentation and can be repositioned as cancer immunotherapeutics to overcome the existing bottlenecks in drug development. Taken together, our results highlight the breadth and magnitude of the proteasome in governing fundamental cellular processes and the immense potential of therapeutics that exploit proteasomes to treat human disease. Citation Format: Priyanka S. Rana, James J. Ignatz-Hoover, James J. Driscoll. HDAC6 inhibition increases proteasome activity and modulates the myeloma immunopeptidome to promote cytotoxic T-cell activity [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C083.

Phenotypic signatures of circulating neoantigen-reactive CD8+ T cells in patients with metastatic cancers

Circulating Tcells from peripheral blood (PBL) can provide a rich and noninvasive source for antitumor Tcells. By single-cell transcriptomic profiling of 36 neoantigen-specific Tcell clones from 6 metastatic cancer patients, we report the transcriptional and cell surface signatures of antitumor PBL-derived CD8+ Tcells (NeoTCRPBL). Comparison of tumor-infiltrating lymphocyte (TIL)- and PBL-neoantigen-specific Tcells revealed that NeoTCRPBL Tcells are low in frequency and display less-dysfunctional memory phenotypes relative to their TIL counterparts. Analysis of 100 antitumor TCR clonotypes indicates that most NeoTCRPBL populations target the same neoantigens as TILs. However, NeoTCRPBL TCR repertoire is only partially shared with TIL. Prediction and testing of NeoTCRPBL signature-derived TCRs from PBL of 6 prospective patients demonstrate high enrichment of clonotypes targeting tumor mutations, a viral oncogene, and patient-derived tumor. Thus, the NeoTCRPBL signature provides an alternative source for identifying antitumor Tcells from PBL of cancer patients, enabling immune monitoring and immunotherapies.

A pilot study of multi-antigen stimulated cell therapy-I plus camrelizumab and apatinib in patients with advanced bone and soft-tissue sarcomas

BackgroundCell-based immunotherapy shows the therapeutic potential in sarcomas, in addition to angiogenesis-targeted tyrosine kinase inhibitor (TKI) and immune checkpoint inhibitor (ICI). Multi-antigen stimulated cell therapy-I (MASCT-I) technology is a sequential immune cell therapy for cancer, which composes of multiple antigen-loaded dendritic cell (DC) vaccines followed by the adoptive transfer of anti-tumor effector T-cells.MethodsIn this phase 1 study, we assessed MASCT-I plus camrelizumab (an ICI against PD-1) and apatinib (a highly selective TKI targeting VEGFR2) in patients with unresectable recurrent or metastatic bone and soft-tissue sarcoma after at least one line of prior systemic therapy. One MASCT-I course consisted of 3 DC subcutaneous injections, followed by 3 active T cell infusions administered 18–27 days after each DC injection. In schedule-I group, 3 DC injections were administered with a 28-day interval in all courses; in schedule-II group, 3 DC injections were administered with a 7-day interval in the first course and with a 28-day interval thereafter. All patients received intravenous camrelizumab 200 mg every 3 weeks and oral apatinib 250 mg daily.ResultsFrom October 30, 2019, to August 12, 2021, 19 patients were enrolled and randomly assigned to schedule-I group (n = 9) and schedule-II group (n = 10). Of the 19 patients, 11 (57.9%) experienced grade 3 or 4 treatment-related adverse events. No treatment-related deaths occurred. Patients in schedule-II group showed similar objective response rate (ORR) with those in schedule-I group (30.0% versus 33.3%) but had higher disease control rate (DCR; 90.0% versus 44.4%) and longer median progression-free survival (PFS; 7.7 versus 4.0 months). For the 13 patients with soft-tissue sarcomas, the ORR was 30.8%, DCR was 76.9%, and median PFS was 12.9 months; for the 6 patients with osteosarcomas, the ORR was 33.3%, the DCR was 50.0%, and median PFS was 5.7 months.ConclusionsOverall, MASCT-I plus camrelizumab and apatinib was safe and showed encouraging efficacy in advanced bone and soft-tissue sarcoma, and schedule-II administration method was recommended.Trial registrationClinicalTrials.gov, NCT04074564.

TRIM29 promotes antitumor immunity through enhancing IGF2BP1 ubiquitination and subsequent PD-L1 downregulation in gastric cancer

Tripartite motif-containing protein 29 (TRIM29) is a member of TRIM family protein which has been reported to play a role in the progress of inflammatory and cancer diseases. However, its specific role in gastric cancer (GC) has yet to be fully understood. Here, we investigated the expression of TRIM29 in gastric cancer and its functions in the antitumor immunity. TRIM29 expression was lower in tumor tissues than that in paired normal tissues. Lower expression of TRIM29 was related to aberrant hypermethylation of CpG islands in TRIM29 gene. Comprehensive proteomics and immunoprecipitation analyses identified IGF2BP1 as TRIM29 interactors. TRIM29 interacted with IGF2BP1 and induced its ubiquitination at Lys440 and Lys450 site by K48-mediated linkage for protein degradation. IGF2BP1 promoted PD-L1 mRNA stability and expression in a 3′UTR and m6A-dependent manner. Functionally, TRIM29 enhanced antitumor T-cell immunity in gastric cancer dependent on the IGF2BP1/PD-L1 axis in vivo and in vitro. Clinical correlation analysis revealed that TRIM29 expression in patient samples was associated with CD8+ immune cell infiltration in the GC microenvironment and the overall survival rates of GC patients. Our findings revealed a crucial role of TRIM29 in regulating the antitumor T-cell immunity in GC. We also suggested that the TRIM29/IGF2BP1/PD-L1 axis could be used as a diagnostic and prognostic marker of gastric cancer and a promising target for GC immunotherapy.

PRMT1 Inhibition Activates the Interferon Pathway to Potentiate Antitumor Immunity and Enhance Checkpoint Blockade Efficacy in Melanoma.

Targeting PRMT1 stimulates interferon signaling by increasing expression of endogenous retroviral elements and double-stranded RNA through repression of DNMT1, which induces antitumor immunity and synergizes with immunotherapy to suppress tumor progression.

Modulatory effects of supplementation of Lentinula edodes mycelia extract and l-arginine on the therapeutic efficacy of immunogenic chemotherapy in colon cancer-bearing mice.

Some chemotherapeutic drugs can induce cancer cell death and enhance antitumor T-cell immunity in cancer-bearing hosts. Immunomodulatory reagents could augment such chemotherapy-induced effects. We previously reported that oral digestion of Lentinula edodes mycelia (L.E.M.) extract or l-arginine supplementation can augment antitumor T-cell responses in cancer-bearing mice. In this study, the effects of L.E.M. extract with or without l-arginine on the therapeutic efficacy of immunogenic chemotherapy by 5-fluorouracil (5-FU)/oxaliplatin (L-OHP) and/or cyclophosphamide (CP) are examined using two mouse colon cancer models. In MC38 and CT26 cancer models, therapy with 5-FU/L-OHP/CP significantly suppressed tumor growth, and supplementation with L.E.M. extract halved the tumor volumes. However, the modulatory effect of L.E.M. extract was not significant. In the CT26 cancer model, supplementation with L.E.M. extract and l-arginine had no clear effect on tumor growth. In contrast, their addition to chemotherapy halved the tumor volumes, although the effect was not significant. There was no difference in the cytotoxicity of tumor-specific cytotoxic T cells generated from CT26-cured mice treated by chemotherapy alone versus chemotherapy combined with L.E.M. extract/ l-arginine. These results indicate that the antitumor effects of immunogenic chemotherapy were too strong to ascertain the effects of supplementation of L.E.M. extract and l-arginine, but these reagents nonetheless have immunomodulatory effects on the therapeutic efficacy of immunogenic chemotherapy in colon cancer-bearing mice.

TMIC-43. THE IMPACT OF MICROGLIA MODULATION ON THE IMMUNE TUMOR MICROENVIRONMENT IN SYNGENEIC GLIOBLASTOMA MOUSE MODELS

Abstract BACKGROUND Glioblastoma is exceptionally resistant to immunotherapies for which the immunosuppressive myeloid compartment is held responsible. As influencer and instructor of T-cell-immunity this compartment mainly consists of Monocyte-derived Cells (MdCs) and tissue-resident (TR) macrophages called microglia (MG). Since the response to immunotherapies depends on antigen presenting cells, we investigate this compartment and its individual role in tumor progression by genetically depleting/activating the TR MG. METHODS We made use of two MG-specific and tamoxifen inducible knockout mouse models in which 1) we activate MG by excision of the activation-repressing transcription factor Sall1 and 2) deplete MG by deleting Csf1r on MG. To investigate the effect of MG activation/depletion on the immune tumor microenvironment (iTME), immunocompetent mice were engrafted with syngeneic glioma cells and the iTME subsequently characterized by scRNAseq, spectral flow cytometry and immunofluorescence imaging. RESULTS Upon Sall1-KO, MG demonstrate a morphologically reactive phenotype. The deletion of Csf1r depletes the brain parenchyma of MG which rapidly gets repopulated by a morphologically distinct microglia-(like) population. Genetic activation of MG leads to a survival benefit in Gl261-engrafted mice compared to Cre- littermates. In contrast, depletion of MG significantly reduces the survival in Gl261 and CT-2A engrafted mice. ScRNAseq confirms the effective depletion of MG and the subsequent emergence of a MG-like cell cluster that transcriptionally remains distinct. The iTME in MG-depleted mice is characterized by a decreased abundance of exhausted and naïve CD8-Tcells and an increased infiltration of MdCs. GSEA demonstrates a dampened inflammatory profile of infiltrating MdCs. CONCLUSION The presence of MG orchestrates the development towards a more antitumorigenic iTME by shaping the antitumoral response of incoming myeloid cells and furthermore directly or indirectly influencing T-cell immunity. Understanding this interplay of MG and MdCs and its impact on instruction of antitumoral T-cell immunity could help guiding the development of novel immunotherapeutic strategies.